The design, synthesis, and anti-tumor mechanism study of N-phosphoryl amino acid modified resveratrol analogues

Article abstract of DOI:10.1016/j.bmc.2008.10.022

A novel series of trans-N-phosphoryl amino acid modified resveratrol analogues were synthesized and evaluated in vitro for their cytotoxic effects against CNE-1 and CNE-2 cell lines. These analogues showed good anti-proliferative activity, among which 8d, 8e, 8j, and 9d displayed much stronger inhibition effect than resveratrol and 8d showed the most potent activity with IC₅₀ value at 3.45 ± 0.82 μM. The anti-tumor effects of 8d, 8e, 8j, and 9d were due to the induction of apoptosis, confirmed by the DNA fragmentation and flow cytometry analysis using PI (propidium iodide) staining and Annexin-V-FITC/PI staining assay. The PI staining assay also showed that 8d, 8e, 8j, and 9d caused cell cycles arrest at G₀-G₁ phase which finally led to cell apoptosis. Further mechanism study on compound 8d against CNE-2 cells has shown the PARP cleavage, which is a hallmark of caspase-3 activation, as well as the activation of caspase-9, and the intracellular ROS generation. These results all suggest that 8d induced a mitochondrial-dependent apoptosis pathway.

Full text of DOI:10.1016/j.bmc.2008.10.022

Bioorganic & Medicinal Chemistry 16 (2008) 10013–10021
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The design, synthesis, and anti-tumor mechanism study of N-phosphoryl
amino acid modified resveratrol analogues
Huachen Liu a,b, Aijun Dong b, Chunmei Gao b, Chunyan Tan b, Hongxia Liu b, Xuyu Zu b, Yuyang Jiang c,b,
*
a Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China
b The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
c School of Medicine, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of trans-N-phosphoryl amino acid modified resveratrol analogues were synthesized and
evaluated in vitro for their cytotoxic effects against CNE-1 and CNE-2 cell lines. These analogues showed
good anti-proliferative activity, among which 8d, 8e, 8j, and 9d displayed much stronger inhibition effect
Received 20 July 2008
Revised 8 October 2008
Accepted 9 October 2008
Available online 12 October 2008
than resveratrol and 8d showed the most potent activity with IC50 value at 3.45 0.82 lM. The anti-
tumor effects of 8d, 8e, 8j, and 9d were due to the induction of apoptosis, confirmed by the DNA fragmen-
tation and flow cytometry analysis using PI (propidium iodide) staining and Annexin-V-FITC/PI staining
assay. The PI staining assay also showed that 8d, 8e, 8j, and 9d caused cell cycles arrest at G0–G1 phase
which finally led to cell apoptosis. Further mechanism study on compound 8d against CNE-2 cells has
shown the PARP cleavage, which is a hallmark of caspase-3 activation, as well as the activation of cas-
pase-9, and the intracellular ROS generation. These results all suggest that 8d induced a mitochon-
drial-dependent apoptosis pathway.
Keywords:
Resveratrol
N-Phosphoryl amino acid
Apoptosis
Mitochondrial pathway
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
compound 2, has been reported to be more potent anti-prolifera-
tive.17 The methoxylation of hydroxyl group may enhance lipophil-
The stilbene moiety has been found commonly in natural prod-
ucts with therapeutically properties. Resveratrol 1 (trans-3,5,40-tri-
hydroxystilbene, Fig. 1), which contains stilbene moiety, is widely
present in nature, specifically in food plants and medicinal
plants.1–5 It has shown a number of therapeutic potential including
anti-inflammatory, cardiovascular diseases, neurodegeneration,
chemoprevention of cancer, and other biological activity.6–9 In par-
ticular, resveratrol showed cancer chemoprevention activity in as-
says representing anti-initiation, anti-promotion, anti-progression
activity, and inhibiting the development of preneoplastic lesions
and tumorigenesis.10 Further evidence has shown that resveratrol
is able to inhibit cell growth and induce apoptosis in various hu-
man cancer cell lines.11–15 Therefore, resveratrol might be consid-
ered as a lead compound for the preparation of analogues with
enhanced bioactivity. In fact, a number of resveratrol analogues
have been synthesized and the structure–activity relationship
(SAR) has been analyzed.11,14,16–21 However, minor attention was
devoted to the lipophilic analogues. pterostilbene 2 (3,5-dime-
thoxy-40-hydroxystilbene, Fig. 1) is a natural methoxylated ana-
logue of resveratrol, which has demonstrated the cancer
chemopreventive activity similar to that of resveratrol.22–24 Com-
pound 3 (40-amino-3,5-dimethoxystilbene, Fig. 1), an analogue of
icity, and thus improve cellular uptake.12,25
On the other hand, phosphorus plays a crucial role in metabo-
lism and N-phosphoryl is considered to be a high biological activity
group.26 Our previous study showed that N-phosphopeptides 4
(Fig. 1) and some N-nitrogen heterocycle compounds can induce
cancer cell apoptosis.27–29 Being intrigued by our previous research,
a series of N-phosphoryl amino acids modified resveratrol ana-
* Corresponding author. Tel./fax: +86 755 2603 2094.
E-mail address: jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
Figure 1. Resveratrol (1), stilbene-related proapoptotic compounds (2–3), and
N-phosphopeptides (4).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.022

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H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
logues were synthesized and their biological activities were evalu-
ated against several human tumor cancer lines in the present study.
played an important role on inhibition activity, suggested by the
stronger inhibition effect of most methoxylated derivatives 8 com-
pared to corresponding non-methoxylated derivatives 9. (2) Most
compounds in 8a–j and 9a–j which contain N-phosphoryl amino
acid moiety showed better inhibition effect than 7a and 7b, which
have the same core structure with amine group only. Combined
with the fact that the best N-phosphoryl amino acid (N-diisopropyl-
2. Result and discussion
2.1. Chemistry
phosphoryl-phenylalanine) itself has IC50 at about 21.34 lM (see
The N-phosphoryl amino acid modified resveratrol analogues
8–9 were synthesized as depicted in Scheme 1. Compound 2a
was prepared by reducing 1a with NaBH4 in high yields, which
was then converted to 3b in the presence of P(Ph)3 and CCl4 under
the reflux condition overnight. Subsequent compound 5, a mixture
of E and Z isomers, were obtained through Wittig reaction.30 These
E/Z mixtures were efficiently converted to trans-stilbene isomer
derivatives 6 with the catalytic amount of I2 in CHCl3. Nitro-deriv-
atives 6 were then reduced by zinc dust in acetic acid to give the
proper amino derivatives 7 in satisfactory yields. Final compounds
8a–j and 9a–j were obtained in high yields through the reaction of
the appropriate N-phosphoryl amino acids with 7a and 7b, respec-
tively, using proper amount of HOBt and DCC in anhydrous THF
solution under nitrogen.
Supporting information), our results indicate the necessity of both
moities, the N-phosphoryl amino acid moiety and the resveratrol
analogues, in order to improve the activity. (3) The cytotoxicity of
both series of compounds 8a–e and 9a–e is relevant to the length
of the alkyl chain on the N-phosphoryl amino acid moiety. Specifi-
cally, the activity decreased as the number of methylene groups in
the chain increased from 1 to 3, suggested by the IC50 values of 8a–c
and 9a–c. Furthermore, 8d–e and 9d–e, which contains 4 and 5
methylene groups in the chain, respectively, showed increased
activity. Especially, compound 8d and 9d showed the most anti-
proliferative potent. (4) Compounds 8f–h and 9f–h have different
substituent groups on a-carbon of amino acid. Our results showed
that the activity of 8f–h and 9f–h decreased as the steric effect in-
creased from methyl, isobutyl, and to isopropyl. This comparison
clearly demonstrates that the steric effect of the amino acid may af-
fect the anti-proliferative potency.
2.2. Anti-proliferative activity of synthesized compounds and
structure–activity relationship
The inhibition of CNE-2 cell growth induced by8d, 8e, 8j, and 9d
at different time intervals is shown in Figure 2. Our results suggest
that not only a dose-dependent but also a time-dependent manner
is involved in the anti-proliferative effects of those compounds.
Two other tumor cells HeLa and K562 were chosen for further
evaluate the anti-proliferation effect of highly active compounds
8d, 8e, 8j, and 9d. Results are shown in Table 2. The fact that the
inhibitory effect of8d, 8e, 8j, and 9d in HeLa and K562 was approx-
imately equivalent to that in CNE suggested that these four com-
pounds could be valuable in cancer treatment.
The newly synthesized compounds 8a–j and 9a–j were initially
evaluated for anti-proliferation activity against human nasopha-
ryngeal carcinoma cell lines CNE-1 and CNE-2 by the MTT assay.
Resveratrol was used as a reference, which has IC50 values at
67.62 and 52.28 lM against CNE-1 and CNE-2 cells, respectively.
As shown in Table 1, most of the compounds displayed good anti-
proliferative activity superior to resveratrol, except 8h, 9h, and 9i.
Several structure–activity relationship was obtained as the follow-
ing: (1) the methoxy groups at 3- and 5-positions of benzene ring
Scheme 1. Reagents and conditions: (a) NaBH4, CH3OH, rt, 2 h; (b) CCl4, PPh3, reflux,12 h; (c) PPh3, CH2Cl2, reflux, 24 h; (d) CH2Cl2, NaH, 4-nitrobenzaldehyde, 0 °C, 24 h; (e)
I2, CHCl3, reflux, 24 h; (f) Zn, CH3COOH, rt, 2 h; (g) HOBt, DCC, THF, rt, 24 h.

H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
10015
Table 1
IC50 of the synthesized compounds of 8(a–j), 9(a–j), 7a, and 7b against of the nasopharyngeal carcinoma (CNE-1 and CNE-2) cell lines IC50
(l
M)a
Compound
CNE-1
IC50
CNE-2
IC50
Compound
CNE-1
IC50
CNE-2
IC50 (lM)
(
l
M)
(
l
M)
(
l
M)
Res.
7a
8a
8b
8c
8d
8e
8f
67.62 4.29
54.49 2.32
8.91 1.18
10.07 1.22
12.33 1.37
5.67 1.15
52.28 4.90
98.67 4.54
7.12 0.77
8.19 1.33
15.15 2.01
3.45 0.82
5.06 1.21
11.72 1.69
14.34 1.77
77.39 6.87
9.83 1.58
9.86 1.67
7b
9a
9b
9c
9d
9e
9f
9g
9h
9i
40.89 2.58
7.50 0.98
29.78 2.77
9.028 1.11
6.36 1.08
8.58 0.95
25.37 3.81
54.26 4.23
49.28 4.78
83.74 8.61
10.08 1.24
65.21 3.15
7.01 1.22
6.81 0.98
6.47 1.14
5.19 1.02
24.32 2.59
12.78 1.76
65.63 7.59
128.44 14.19
92.06 6.29
12.19 1.33
6.37 1.31
29.17 4.61
55.27 8.94
116.10 11.84
43.42 5.51
8.11 0.84
8g
8h
8i
8j
9j
a
Results were obtained after 72 h of treatment and means SEM of three experiments. Differences were considered significant at P < 0.05.
Figure 2. Time dependence of IC50 values of compound 8d, 8e, 8j, and 9d against
CNE-2 cell lines. Cells were treated with the corresponding compound at 24, 48, and
72 h.
Figure 3. Apoptosis was evaluated by the induction of DNA fragmentation. CNE-2
cells were treated with compound 8d, 8e, 8j, and 9d and resveratrol at 50 lM for
24 h. Untreated CNE-2 cells were used as control.
Table 2
IC50 values of the synthesized compounds of 8d, 8e, 8j, and 9d against HeLa and K562
cell linesa
apoptosis is the occurrence of a sub-G0–G1 cell population by flow
cytometry analysis of DNA-content histogram after propidium io-
8d
8e
8j
9d
dide (PI) staining. As shown in Figure 4, cells treated with 50 lM
HeLa
K562
5.62 0.95
5.32 1.02
6.41 1.45
6.89 1.63
9.53 2.12
9.15 1.51
9.75 1.20
9.58 1.62
8d, 8e, 8j, and 9d for 24 h resulted in the obvious sub-G0–G1 peak,
ranged from 32.15% to 91.59%. From Figure 4 we also find that cell
cycle was arrested in G0–G1 phase in this condition.
a
Results were obtained after 72 h of treatment and means SEM of three
experiments. Differences were considered significant at P < 0.05.
In addition, the Annexin-V/PI binding assay was used to further
confirm the drug induced apoptosis effect. Annexin-V conjugated
with the fluorochrome FITC serves as a marker for apoptotic cells
because it has a strong binding affinity to phosphatidylserine
(PS), which re-distributes from the inner to the outer layer of the
plasma membrane in apoptotic cells. Figure 5 shows the histogram
with dual parameters of Annexin-V-FITC and PI, among which the
upper right represents the late stage apoptotic cells (Annexin posi-
tive and PI positive), and the lower right represents the early stage
apoptotic cell population (Annexin positive and PI negative). As the
2.3. Mechanism of the anti-proliferative activity
It was reported that resveratrol can induce apoptosis in many
tumor cell lines.12 We therefore speculate that these novel resvera-
trol analogues may also have similar effects. On the basis of the
cytotoxicity study, four compounds, 8d, 8e, 8j, and 9d, were se-
lected for further examinations to determine whether the cytotox-
icity is mediated by the induction of apoptosis. Apoptosis is
regulated by genetic mechanisms and is principally characterized
by morphological and biochemical changes in the nucleus, includ-
ing chromatin condensation and internucleosomal DNA fragmen-
tation. As we expected, the exposure of CNE-2 cells to compound
8d, 8e, 8j, and 9d for 24 h resulted in the appearance of DNA lad-
ders on the agarose gels, which is a hallmark of apoptosis, as
shown in Figure 3. Another typical feature of cells undergoing
concentration of 8d increased from 5 to 50 lM, the Annexin-V po-
sitive/PI negative cells increased from 6.45% to 21.24%, and then
decreased back to 18.78%, whereas the double positive cells in-
creased from 9.86% to 66.79%, suggesting that more and more
apoptotic cells progressed from the early stage to the late stage
resulting in either death or secondary necrosis under the effect of
8d at higher concentrations. This confirms again that 8d induced
the cell apoptosis of CNE-2 cells.

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H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
2.4.3. Compound induces the transient generation of ROS
In a mitochondrial-dependent apoptosis, mitochondria undergo
what is referred to as the mitochondrial permeability transition
(MPT), which has several consequences including the collapse of
D
Wm, Ca2+ release, and the generation of reactive oxygen species
(ROS). To further confirm that the mitochondria pathway was acti-
vated in the compound 8d induced apoptotic process, we traced
the ROS level in the treated CNE-2 cells. As shown inFigure 8, com-
pound 8d rapidly induced the generation of ROS and the percent-
age of cells with higher fluorescence reached a maximum at 4 h
and then declined, reached the minimum value at 19 h determined
by DCFH-DA fluorescence.34
3. Conclusion
In conclusions, combination of resveratrol and N-phosphoryl
amino acids offered the discovery of a series of potent growth
inhibitors of CNE-1 and CNE-2 cell lines. Compound 8d has the
most potent inhibitory activity, which is 15-fold higher than that
of resveratrol. Compounds 8d, 8e, 8j, and 9d induced cell cycle
G0/G1 arrest and apoptosis in a dose- and time-dependent manner.
Compound 8d induced apoptosis was associated with caspase-9
activation, indicating that the mitochondrial pathway was in-
volved in the apoptosis pathway, which was further confirmed
by the generation of ROS. Taking into consideration that these no-
vel compounds possess anti-tumor activity, they may become can-
didates of effective chemopreventive agents for cancer
chemotherapy.
4. Experimental
4.1. Chemistry
Figure 4. Flow cytometry analysis of cell membrane integrity (PI staining) and cell
cycle arrest. CNE-2 cells were treated with compound 8d, 8e, 8j, and 9d at 50 lM for
24 h. The percentage of apoptotic events accumulating under the marker in the
Sub-G0–G1 phase and a decrease of cells in both S and G2/M phases, especially in G2/
M of the cell cycle was analyzed using CellQuest software (Becton–Dickinson).
1H NMR and 13C NMR were recorded in the indicated deuter-
ated solvent at 300 or 500 MHz on Bruker NMR spectrometers.
Chemical shifts are expressed in ppm (d) and coupling constants
J in Hertz (Hz). High-resolution mass spectra were obtained with
Waters micromass Q-Tof Premier mass spectrometer. Melting
points were determined by the open capillary method on a SGW
X-4 electrothermal melting apparatus and are uncorrected. Reac-
tion progress was monitored using analytical thin-layer chroma-
tography (TLC) on pre-coated silica gel plates and the spots were
detected under UV light (254 nm). All other reagents and analytical
grade solvents were bought from commercial sources and used
without further purification unless indicated. Methylene chloride
(CH2Cl2) was distilled from calcium hydride (CaH2) and tetrahydro-
furan (THF) was distilled from sodium immediately prior to use.
2.4. Apoptosis pathway study
2.4.1. Activation of caspase-3 activity
Apoptosis is mainly brought about by activation of caspases, a
family of cystein proteases capable of cleaving essential cellular
substrates after aspartic residues. Caspase-3 is the key apoptotic
executive protein which could be activated by both death-receptor
pathway and mitochondrial pathway. We tested the induction of
poly-(ADP-ribose)-polymerase (PARP) cleavage by the four ana-
logues (8d, 8e, 8j, and 9d). The 89 kDa band, which is an evidence
of PARP cleavage, was observed. Since PARP is a substrate for cas-
pase-3, the apoptotic effect of compounds might be caspase-med-
iated (Fig. 6).31,32
4.1.1. Synthesis of 3,5-dimethoxylbenzyl alcohol (2)
The NaBH4 (35 mmol) of methanol solution was dropped into a
solution of 10 mmol of 3,5-dimethoxybenzaldehyde at room tem-
perature, keeping the dropped in an appropriative velocity. The
mixture was stirred for 2 h and evaporated methanol under vac-
uum, then adding water, extracted with 20 ml EtOAc twice, the or-
ganic layer was dried over anhydrous NaSO4, and evaporated under
vacuum obtaining the compound 2. Yield 94%, mp: 47–48 °C, 1H
NMR (CDCl3, 500 MHz): d 3.80 (s, 6H), 4.52 (s, 2H), 6.41 (m, 1H),
6.54 (m, 2H).
2.4.2. Activation of caspase-8 or caspase-9?
To gain insights into the pathway by which caspase-3 was
activated, we investigated the activity of caspase-8 and cas-
pase-9. As known, caspase-8 is involved in the death-receptor
pathway, while caspase-9 mediates the mitochondrial path-
way.33 Once activated, both caspase-8 and caspase-9 can then
activate downstream caspase-3, which finally causes cell apopto-
sis. Figure 7 demonstrated that the activity of caspase-9 in-
creased dramatically in the 8d-treated CNE-2 cells after
different length of incubation times ranging from 0 to 24 h.
While on the contrary, the activity of caspase-8 had no obvious
change. This comparison suggests that compound 8d had no sig-
nificant effect on the activation of caspase-8. Therefore, com-
pound 8d induces CNE-2 cell apoptosis through mitochondria
pathway instead of the death-receptor pathway.
4.1.2. Synthesis of 3,5-dimethoxylbenzyl chloride (3)
A solution of 3,5-dimethoxylbenzyl alcohol (10 mmol) and PPh3
(11 mmol) in CCl4 (50 ml) was stirred at reflux condition. After
12 h the solvent of CCl4 was evaporated under vacuum, and the
mixture was filtered through silica gel/Celite to afford 3,5-dim-
ethoxylbenzyl chloride as a white solid. (92%, mp: 46–48 °C), 1H

H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
10017
Figure 5. Flow cytometric analysis of phosphoatidylserine externalization (Annexin-V binding) and cell membrane integrity (PI staining). CNE-2 cells were treated with
compound 8d at 5, 10, 25, and 50 M for 24 h.
l
Figure 6. The proapoptotic effect was determined by the PARP cleavage assay,
evaluated as appearance of the 89 kDa band in CNE-2 cells treated with 8d, 8e, 8j,
and 9d at 50 lM for 24 h.
Figure 8. The ROS generation determined in terms of DCFH-DA fluorescence by
flow cytometric analysis. CNE-2 cells was treated with compound 8d at 50 lM for
the indicated times.
NMR (CDCl3, 500 MHz): d 3.80 (s, 6H), 4.52 (s, 2H), 6.41 (m, 1H),
6.54 (m, 2H).
4.1.3. Synthesis of (E/Z)-40-nitrostilbene derivatives (5)
The appropriate benzyl chloride (10 mmol) was dissolved in
CH2Cl2 (50 ml) and then PPh3 (13 mmol) was added. The mix-
ture was stirred for 24 h at room temperature and then cooled
in ice-water bath, and then 4-nitrobenzaldehyde (10 mmol) was
added. The solution of NaH in CH2Cl2 was dropped with appro-
Figure 7. Caspase-8 and capase-9 activities were detected by Ac-IETD-pNA and Ac-
LEHD-pNA at 0, 2, 4, 8, 14, and 24 h of compound 8d at 50 lM, respectively. Results
are mean SD (n = 3).
priated velocity into the reaction system under a nitrogen

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H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
atmosphere. After 24 h, water was added, and the product was
isolated by extraction with CH2Cl2. The organic phase was
washed with saturated brine and dried over anhydrous NaSO4.
The solvent was removed under reduced pressure to obtain 5
(E/Z) as a mixture of isomers which was determined by 1H
NMR signals.
4.1.6.2. Compound 8b: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-b-alanine)amido-E-stilbene. Yield: 66%; mp:
153–154 °C; 31P NMR (CDCl3, 202 MHz): d 7.070. 1H NMR (CDCl3,
500 MHz): d 1.29 (m, 12H), 2.04 (m, 1H), 2.63 (m, 2H), 3.31 (m,
2H), 3.82 (s, 6H), 4.59 (m, 2H), 6.38 (m, 1H), 6.65 (m, 2H), 6.95
(d, 1H, J13 = 16.50 Hz), 7.03 (d, 1H, J13 = 16.50 Hz), 7.43 (d, 2H,
J13 = 8.50 Hz), 7.60 (d, 2H, J13 = 8.50 Hz), 8.85 (s, 1H). 13C NMR
(CDCl3, 125 MHz): d 23.77, 23.81, 37.99, 38.93, 55.34, 71.27,
71.32, 99.98, 104.54, 119.90, 127.07, 132.98, 137.98, 139.50,
161.03, 127.72, 128.65, 169.97. HRMS: calcd for [C25H36N2O6P]+:
491.2311; found: 491.2331.
4.1.4. Transform 5 (E/Z) to trans-4-nitrostilbene (6a) or trans-
3,5-dimethoxyl-40-nitrostilbene (6b)
A solution of 5 (E/Z) (10 mmol) in CHCl3 (60 ml) with the cat-
alytic amount of I2 was stirred and reflowed for 12 h, the catalytic
I2 of system reaction was reduced with saturated sodium hydro-
gen sulfite, and continued stirring for 2 h. The production was
extracted with 30 ml CHCl3 twice. The organic phase was washed
with saturated brine and dried over NaSO4 and evaporated under
vacuum to obtain compound 6a–b. Compound 6a: 95%, mp: 157–
159 °C. 1H NMR (DMSO, 500 MHz,): d 6.55 (m, 2H), 6.88 (d, 1H,
J13 = 16.40 Hz), 7.05 (d, 1H, J13 = 16.40 Hz), 7.17 (m, 1H), 7.26
(m, 2H), 7.31 (m, 2H), 7.48 (m, 2H). Compound 6b: 96%, mp:
135–137 °C. 1H NMR (CDCl3, 300 MHz): d 3.85 (s, 6H), 6.46 (m,
1H), 6.70 (m, 2H), 7.11 (d, 1H, J13 = 16.20 Hz), 7.21 (d, 1 H,
J13 = 16.20 Hz), 7.63 (d, 2H, J13 = 8.70 Hz), 8.23 (d, 2H,
J13 = 9.00 Hz).
4.1.6.3. Compound 8c: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-4-aminobutyric)amido-E-stilbene.
Yield: 70%;
mp: 109–111 °C; 31P NMR (CDCl3, 202 MHz): d 8.40. 1H NMR
(CDCl3, 500 MHz): d 1.30 (m, 12H), 1.92 (m, 2H), 2.54 (m, 2H),
2.99 (m, 2H), 3.82 (s, 6H), 4.59 (m, 2H), 6.38 (m, 1H), 6.65 (m,
2H), 6.95 (d, 1H, J13 = 16.50 Hz), 7.04 (d, 1H, J13 = 16.50 Hz), 7.43
(d, 2H, J13 = 8.50 Hz), 7.66 (d, 2H, J13 = 8.50 Hz), 9.44 (s, 1H). 13C
NMR (CDCl3, 125 MHz): d 23.78, 23.81, 28.10, 33.98, 39.81, 55.36,
71.30, 71.34, 99.95, 104.50, 119.90, 127.00, 132.72, 138.44,
139.57, 161.03, 127.50, 128.78, 171.59. HRMS: calcd for
[C26H38N2O6P]+: 505.2468; found: 505.2476.
4.1.5. Synthesis of trans-4-aminostilbene and trans-3,5-dimeth-
oxyl-40-aminostilbene (7a–b)
4.1.6.4. Compound 8d: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-5-aminovaleric)amido-E-stilbene.
Yield 63%;
To a stirred solution of 6a–b (10 mmol) in acetic acid was added
zinc dust (40 mmol). After 2 h, the solution was filtered under vac-
uum through silica gel and the filtrate was concentrated under vac-
uum. The product was separated by silica gel/Celite to obtain
compounds 7a–b. Compound 7a: 74%, 1H NMR (CDCl3,
300 MHz,): d 3.82 (s, 6H), 6.36 (m, 1H), 6.66 (m, 4H), 6.85 (d, 1H,
J13 = 16.20 Hz), 7.00 (d, 1H, J13 = 16.20 Hz), 7.33 (m, 2H). 7b:
73.4%, mp: 88–91 °C. 1H NMR (CDCl3, 300 MHz,): d 3.75 (s, 2H),
6.68 (d, 2H), 6.92 (d, 1H, J13 = 16.20 Hz), 7.03 (d, 1H,
J13 = 16.20 Hz), 7.21 (m, 1H), 7.32 (m, 2H), 7.34 (m, 2H), 7.47 (m,
2H).
oil; 31P NMR (CDCl3, 202 MHz): 7.32. 1H NMR (CDCl3,
d
500 MHz): d 1.31 (m,12H), 1.57 (m, 2H), 1.84 (m, 2H), 2.40 (m,
2H), 2.61 (m, 1H), 3.00 (m, 2H), 3.82 (s, 6H), 4.58 (m, 2H),
6.38 (m, 1H), 6.65 (d, 2H), 6.95 (d, 1H, J13 = 16.50 Hz), 7.03 (d,
1H, J13 = 16.50 Hz), 7.44 (d, 2H, J13 = 8.50 Hz),7.60 (d, 2H,
J13 = 8.50 Hz), 8.51 (s, 1H). 13C NMR (CDCl3, 125 MHz): d 22.51,
23.78, 23.81, 30.88, 36.67, 40.44, 55.33, 70.96, 71.00, 99.86,
104.43, 119.90, 127.01, 132.60, 138.41, 139.50, 160.96, 127.44,
128.70, 171.92. HRMS: calcd for [C27H40N2O6P]+: 519.2426;
found: 519.2635.
4.1.6.5. Compound 8e: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
4.1.6. General procedure for synthesis of compound 8a–j and
9a–j
phosphoryl-6-aminocaproic)amido-E-stilbene.
Yield 61%;
mp: 80–82 °C; 31P NMR (CDCl3, 202 MHz): d 7.59. 1H NMR (CDCl3,
500 MHz): d 1.30 (m, 12H), 1.40 (m, 2H), 1.53 (m, 2H), 1.74 (m, 2H),
2.38 (m, 2H), 2.63 (m, 1H), 2.90 (m, 2H),3.82 (s, 6H), 4.57 (m, 2H),
6.38 (m, 1H), 6.65 (m, 2H), 6.95 (d, 1H, J13 = 16.50 Hz), 7.03 (d, 1H,
J13 = 16.50 Hz), 7.43 (d, 2H, J13 = 8.50 Hz), 7.59 (d, 2H, J13 = 8.50 Hz),
8.32 (s, 1H). 13C NMR (CDCl3, 75 MHz): d 23.81, 23.86, 25.08, 26.08,
31.25, 37.31, 41.17, 55.36, 70.78, 70.86, 99.86, 104.41, 119.87,
127.08, 132.76, 138.09, 139.45, 160.95, 127.55, 128.63, 171.61.
HRMS: calcd for [C28H42N2O6P]+: 533.2781; found: 533.2770.
The appropriate N-phosphoryl amino acids (1.05 mmol), the
compounds 7a–b (1 mmol) and HOBt (1.05 mmol) in anhydrous
tetrahydrofuran was stirred at under ice-water bath in nitrogen
atmosphere, and then the solution of DCC (1.05 mmol) in THF
(20 ml) was dropped with a appropriated velocity. The tempera-
ture of system reaction was slowly heightened to room tempera-
ture. After 24 h, the deposition was filtered and the filtrate was
concentrated under vacuum. The residue was washed with
saturated NaHCO3 and stirred. The aqueous solution was twice
extracted with 30 ml CHCl3. The combined organic phases were
washed with saturated NaHCO3, 10% citric acid solution and
saturated brine by turns twice, dried over anhydrous NaSO4, and
evaporated under vacuum. The products were separated by silica
gel/Celite to obtain compound 8a–j and 9a–j (eluent, chloroform/
methanol).
4.1.6.6. Compound 8f: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-
L
-Ala)amido-E-stilbene.
Yield 73%; mp: 135–
137 °C; 31P NMR (CDCl3, 202 MHz): d 5.86. 1H NMR (CDCl3,
500 MHz): d 1.33 (m, 12H), 1.49 (d, 3H, J13 = 6.80 Hz), 3.66 (m,
1H), 3.82 (s, 6H), 4.02 (m, 1H), 4.61 (m, 2H), 6.38 (m, 1H), 6.65
(m, 2H), 6.96 (d, 1H, J13 = 16.50 Hz), 7.04 (d, 1H, J13 = 16.50 Hz),
7.45 (d, 2H, J13 = 8.50 Hz), 7.63 (d, 2H, J13 = 8.50 Hz), 9.43 (s, 1H).
13C NMR (CDCl3, 75 MHz): d 21.10, 23.74, 23.79, 52.13, 55.38,
71.65, 71.78, 99.86, 104.41, 119.81, 127.08, 132.89, 138.04,
139.45, 160.95, 127.62, 128.63, 172.13. HRMS: calcd for
[C25H36N2O6P]+: 491.2311; found: 491.2295.
4.1.6.1. Compound 8a: 3,5-dimethoxyl-40-(N-O,O0-diisopropylphos-
phoryl-
L
-Gly)amido-E-stilbene.
Yield 71%; mp: 118–120 °C;
31P NMR (CDCl3, 202 MHz): d 6.646. 1H NMR (CDCl3, 500 MHz): d 1.35
(d, 12H, J13 = 6.20 Hz), 3.46 (m, 1H), 3.75 (d, 2H, J13 = 13.10 Hz), 3.83
(s, 6H), 4.66 (m, 2H), 6.39 (m, 1H), 6.65 (m, 2H), 6.97 (d, 1H,
J13 = 16.25 Hz), 7.05 (d, 1H, J13 = 16.25 Hz), 7.47 (d, 2H, J13 = 8.50 Hz),
7.58 (d, 2H, J13 = 8.50 Hz), 8.95 (s,1H); 13C NMR (CDCl3, 125 MHz,): d
23.77, 45.77, 55.35, 71.96, 71.92, 99.98, 104.55, 119.96, 127.16,
133.25, 137.51, 139.45, 161.03, 127.89, 128.57, 168.53. HRMS: calcd
for [C24H34N2O6P]+: 477.2155; found: 477.2168.
4.1.6.7. Compound 8g: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-
L
-Leu)amido-E-stilbene.
Yield 69%; mp: 169–
173 °C; 31P NMR (CDCl3, 202 MHz): d 6.27. 1H NMR (CDCl3,
500 MHz): d 0.97 (d, 6H, J13 = 6.25 Hz), 1.30 (m, 12H), 1.57 (m,
1H), 1.80 (m, 2H), 3.34 (m,1H), 3.83 (s, 6H), 3.87 (m, 1H), 4.60

H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
10019
(m, 2H), 6.39 (m, 1H), 6.65 (m, 2H), 6.96 (d, 1H, J13 = 16.50 Hz), 7.04
(d, 1H, J13 = 16.50 Hz), 7.45 (d, 2H, J13 = 8.50 Hz), 7.62 (d, 2H,
J13 = 8.50 Hz), 9.24 (s, 1H). 13C NMR (CDCl3, 75 MHz): d 21.99,
23.03, 23.73, 23.77, 24.52, 43.79, 55.17, 55.35, 71.71, 71.79,
99.85, 104.40, 119.88, 126.99, 132.76, 138.24, 139.48, 160.96,
127.51, 128.70, 172.574. HRMS: calcd for [C28H42N2O6P]+:
533.2781; found: 533.2795.
4.1.6.13. Compound 9c: 4-(N-O,O0-diisopropylphosphoryl-4-
aminobutyric)amido-E-stilbene. Yield 63%; mp: 115–118 °C;
31P NMR (CDCl3, 202 MHz): d 8.22. 1H NMR (CDCl3, 500 MHz): d
1.31 (m, 12H), 1.91 (m, 2H), 2.54 (m, 2H), 3.01 (m, 2H), 4.60 (m,
2H), 7.02 (d, 1H, J13 = 16.50 Hz), 7.07 (d, 1H, J13 = 16.50 Hz), 7.34
(m, 2H), 7.45 (m, 2H), 7.49 (m, 2H), 7.67 (m, 2H), 9.31 (s, 1H).
13C NMR (CDCl3, 125 MHz): d 23.76, 23.78, 27.87, 33.97, 39.98,
71.10, 71.14, 120.01, 127.36, 127.52, 128.26, 128.62, 132.89,
137.51, 138.43, 126.35, 126.89, 171.71. HRMS: calcd for
[C24H34N2O4P]+: 445.2256; found: 445.2261.
4.1.6.8. Compound 8h: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-
L-Val)amido-E-stilbene.
Yield 71%; mp: 170–
172 °C; 31P NMR (CDCl3, 202 MHz): d 6.18. 1H NMR (CDCl3,
500 MHz): d 0.99 (d, 3H, J13 = 6.80 Hz), 1.30 (m, 12H), 2.28 (m,
1H), 3.50 (m, 1H), 3.72 (m, 1H), 3.83 (s, 6H), 4.61 (m, 2H), 6.39
(m, 1H), 6.65 (m, 2H), 6.96 (d, 1H, J13 = 16.50 Hz), 7.04 (d, 1H,
J13 = 16.50 Hz), 7.45 (d, 2H, J13 = 8.50 Hz), 7.63 (d, 2H,
J13 = 8.50 Hz), 9.17 (s, 1H). 13C NMR (CDCl3, 125 MHz): d 19.82,
21.36, 25.64, 25.71, 33.79, 57.26, 64.06, 73.46, 73.58, 101.88,
106.44, 121.87, 128.9, 134.87, 139.88, 141.44, 162.95, 129.58,
130.62, 173.34. HRMS: calcd for [C27H40N2O6P]+: 519.2624; found:
519.2617.
4.1.6.14. Compound 9d: 4-(N-O,O0-diisopropylphosphoryl-5-
aminovaleric)amido-E-stilbene.
Yield 62%; mp: 122–125 °C;
31P NMR (CDCl3, 202 MHz): d 7.969. 1H NMR (CDCl3, 500 MHz): d
1.31 (m, 12H), 1.57 (m, 2H), 1.85 (m, 2H), 2.40 (m, 2H), 3.00 (m,
2H), 4.58 (m, 2H), 7.02 (d, 1H, J13 = 16.50 Hz), 7.06 (d, 1H,
J13 = 16.50 Hz), 7.25 (m, 1H), 7.34 (m, 2H), 7.45 (d, 2H), 7.49 (m,
2H), 7.61 (m, 2H), 8.43 (s, 1H). 13C NMR (CDCl3, 75 MHz): d
22.53, 23.78, 23.84, 30.79, 36.72, 40.32, 71.01, 71.09, 119.86,
127.40, 127.46, 128.17, 128.66, 132.80, 137.44, 138.29, 126.35,
126.96, 171.93. HRMS: calcd for [C25H36N2O4P]+: 459.2413; found:
459.2420.
4.1.6.9. Compound 8i: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-
L-Phe)amido-E-stilbene.
Yield
75%;
165–
167 °C; 1H NMR (CDCl3, 500 MHz): d 1.26 (m, 12H), 3.14 (m, 2H),
3.47 (m, 1H), 3.82 (s, 6H), 4.11 (m, 1H), 4.49 (m, 2H), 6.41 (m,
1H), 6.66 (m, 2H), 6.97 (d, 1H, J13 = 16.50 Hz), 7.04 (d, 1H,
J13 = 16.50 Hz), 7.25 (m, 5H), 7.45 (d, 2H, J13 = 8.50 Hz), 7.51 (d,
2H, J13 = 8.50 Hz), 8.63 (s, 1H). 13C NMR (CDCl3, 75 MHz): d 23.72,
23.78, 39.97, 57.70, 71.77, 71.85, 99.88, 104.44, 120.00, 126.98,
127.07, 128.48, 129.68, 133.12, 136.69, 137.54, 139.43, 160.97,
127.75, 128.60, 170.95. HRMS: calcd for [C31H40N2O6P]+:
567.2624; found: 567.2622.
4.1.6.15. Compound 9e: 4-(N-O,O0-diisopropylphosphoryl-6-
aminocaproic)amido-E-stilbene.
Yield 66%; mp: 127–128 °C;
31P NMR (CDCl3, 202 MHz): d 7.61. 1H NMR (CDCl3, 500 MHz): d
1.31 (m, 12H), 1.43 (m, 2H), 1.56 (m, 2H), 1.76 (m, 2H), 2.38 (m,
2H), 2.50(m, 1H), 2.92 (m, 2H), 4.58 (m, 2H), 7.02 (d, 1H,
J13 = 16.50 Hz), 7.07 (d, 1H, J13 = 16.50 Hz), 7.23 (m, 1H), 7.34 (m,
2H), 7.46 (m, 2H), 7.49 (m, 2H), 7.56 (m, 2H), 7.77 (s, 1H). 13C
NMR (CDCl3, 75 MHz): d 23.80, 23.86, 25.10, 26.09, 31.23, 37.37,
41.15, 70.81, 70.88, 119.90, 127.44, 127.60, 128.11, 128.67,
133.01, 137.41, 137.96, 126.37, 127.00, 171.56. HRMS: calcd for
[C26H38N2O4P]+: 473.2569; found: 473.2581.
4.1.6.10. Compound 8j: 3,5-dimethoxyl-40-(N-O,O0-diisopropyl-
phosphoryl-
L-Pro)amido-E-stilbene.
Yield 73%; oil; 31P NMR
(CDCl3, 202 MHz): d 7.82. 1H NMR (CDCl3, 500 MHz): d 1.32 (m,
12H), 1.95 (m, 4H), 2.53 (m, 1H), 3.24 (m, 2H), 3.83 (s, 6H), 4.27
(m, 1H), 4.62 (m, 2H), 6.39 (m, 1H), 6.65 (m, 2H), 6.96 (d, 1H,
J13 = 16.50 Hz), 7.04 (d, 1H, J13 = 16.50 Hz), 7.46 (d, 2H,
J13 = 8.50 Hz), 7.59 (d, 2H, J13 = 8.50 Hz), 9.60 (s, 1H). 13C NMR
(CDCl3, 75 MHz): d 23.92, 23.98, 25.58, 29.00, 47.81, 61.73, 55.37,
71.78, 71.86, 99.87, 104.39, 119.45, 127.18, 132.79, 137.98,
139.51, 160.95, 27.54, 128.67, 170.81. HRMS: calcd for
[C27H38N2O6P]+: 517.2468; found: 517.2477.
4.1.6.16. Compound 9f: 4-(N-O,O0-diisopropylphosphoryl-
mido-E-stilbene.
Yield 70%; mp: 155–157 °C; 31P NMR (CDCl3,
L-Ala)a-
202 MHz): d 5.13. 1H NMR (CDCl3, 300 MHz): d 1.32 (m, 12H), 1.50
(d, 3H, J13 = 6.85 Hz), 3.87 (m, 1H), 4.05 (m, 1H), 4.61 (m, 2H), 7.02
(d, 1H, J13 = 16.50 Hz), 7.08 (d, 1H, J13 = 16.50 Hz), 7.23 (m, 1H),
7.34 (m, 2H), 7.47 (m, 4H),7.65 (m, 2H), 9.58 (s, 1H). 13C NMR (CDCl3,
75 MHz): d 21.20, 23.77, 23.80, 52.17, 71.71, 71.79, 119.85, 127.46,
127.65, 128.13, 128.69, 133.09, 137.42, 137.99, 126.39, 127.01,
172.21. HRMS: calcd for [C23H32N2O4P]+: 431.2100; found: 431.2107.
4.1.6.11. Compound 9a: 4-(N-O,O0-diisopropylphosphoryl-
amido-E-stilbene.
Yield 61.8%; mp: 139–141 °C; 31P NMR
L-Gly)
4.1.6.17. Compound 9g: 4-(N-O,O0-diisopropylphosphoryl-
Leu)amido-E-stilbene.
Yield 76%; mp: 168–170 °C; 31P
L-
(CDCl3, 202 MHz): d 5.91. 1H NMR (CDCl3, 500 MHz): d 1.33 (m,
12H), 3.77 (m, 2H), 3.90 (m, 1H), 4.64 (m, 2H), 7.03 (d, 1H,
J13 = 16.50 Hz), 7.06 (d, 1H, J13 = 16.50 Hz), 7.24 (m, 1H), 7.34 (m,
2H),7.46 (m, 2H), 7.48 (m, 2H), 7.59 (m, 2H), 9.19 (s, 1H). 13C NMR
(CDCl3, 125 MHz): d 23.77, 23.81, 45.80, 71.90, 71.94, 119.83,
127.47, 127.94, 128.08, 128.65, 133.48, 137.39, 137.44, 126.40,
127.08, 170.29. HRMS: calcd for [C22H30N2O4P]+: 417.1943; found:
417.1951.
NMR (CDCl3, 202 MHz): d 5.83. 1H NMR (CDCl3, 500 MHz): d 0.97
(d, 6H, J13 = 6.35 Hz), 1.30 (m, 12H), 1.58 (m, 1H), 1.80 (m, 2H),
3.48 (m, 1H), 3.88 (m, 1H), 4.62 (m,2H), 7.03 (d, 1H,
J13 = 16.50 Hz), 7.07 (d, 1H, J13 = 16.50 Hz), 7.23 (m, 1H), 7.34 (m,
2H), 7.46 (m, 2H), 7.49 (m, 2H), 7.63 (m, 2H), 9.31 (s, 1H). 13C
NMR (CDCl3, 75 MHz): d 21.97, 23.04, 23.73, 23.80, 24.52, 43.59,
55.22, 71.75, 1.83, 119.86, 127.43, 127.58, 128.17, 128.68, 133.03,
137.45, 138.05, 126.37, 126.94, 172.37. HRMS: calcd for
[C26H38N2O4P]+: 473.2569; found: 473.2584.
4.1.6.12. Compound 9b: 4-(N-O,O0-diisopropylphosphoryl-b-ala-
nine)amido-E-stilbene.
Yield 57.6%; mp: 172–174 °C; 31P
4.1.6.18. Compound 9h: 4-(N-O,O0-diisopropylphosphoryl-
Val)amido-E-stilbene.
L-
NMR (CDCl3, 202 MHz): d 6.70. 1H NMR (CDCl3, 300 MHz): d 1.30
(m, 12H), 2.63 (m, 2H), 3.32 (m, 2H), 4.60 (m, 2H), 7.02 (d, 1H,
J13 = 16.50 Hz), 7.07 (d, 1H, J13 = 16.50 Hz), 7.22 (m, 1H), 7.35 (m,
2H), 7.45 (m, 2H), 7.49 (m, 2H), 7.60 (m, 2H), 8.61 (s, 1H). 13C
NMR (CDCl3, 125 MHz): d 23.80, 23.84, 38.05, 38.72, 71.17, 71.22,
120.02, 127.42, 127.63, 128.16, 128.66, 133.13, 137.45, 138.13,
126.37, 126.96, 170.29. HRMS: calcd for [C23H32N2O4P]+:
431.2100; found: 431.2107.
Yield 74%; mp: 140–142 °C; 31P NMR
(CDCl3, 202 MHz): d 6.39. 1H NMR (CDCl3, 500 MHz): d 0.99 (d,
3H, J13 = 6.80 Hz), 1.05 (d, 3H, J13 = 6.80 Hz), 1.31 (m, 12H), 2.29
(m, 1H), 3.46 (m, 1H), 3.68 (m, 1H), 4.61 (m, 2H), 7.03 (d, 1H,
J13 = 16.50 Hz), 7.08 (d, 1H, J13 = 16.50 Hz), 7.24 (m, 1H), 7.35 (m,
2H), 7.46 (m, 2H), 7.50 (m, 2H), 7.62 (d, 2H), 9.09 (s, 1H). 13C
NMR (CDCl3, 75 MHz): d 17.87, 19.43, 23.69, 23.74, 31.89, 61.96,

10020
H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
71.99, 72.07, 119.85, 127.46, 127.65, 128.13, 128.69, 133.09,
137.42, 137.99, 126.39, 127.01, 174.11. HRMS: calcd for
[C25H36N2O4P]+: 459.2413; found: 459.2419.
70% ethanol at 4 °C overnight, and resuspended at 5 ꢀ 105/ml cells
of propidium iodide [50 lg/ml in 0.1% sodium citrate plus 0.03% (v/
v) Nonidet P-40] and incubated at ice bath for 30 min the tubes
were packed with tin foil at ice bath until use in flow cytometry
analysis using an FACscan flow cytometry (Becton–Dickinson) with
CellQuest software. Surface exposure of phosphatidylserine in
apoptosis was detected by detection kit.
4.1.6.19. Compound 9i: 4-(N-O,O0-diisopropylphosphoryl-
Phe)amido-E-stilbene.
Yield 73%; mp: 188–191 °C; 31P NMR
L-
(CDCl3, 202 MHz): d 5.54. 1H NMR (CDCl3, 300 MHz): d 1.26 (m,
12H), 3.11 (m, 2H), 3.48 (m, 1H), 4.11 (m, 1H), 4.49 (m, 2H), 7.06
(m, 2H), 7.26 (m, 6H), 7.35 (m, 2H), 7.50 (m, 6H), 8.58 (s, 1H). 13C
NMR (CDCl3, 75 MHz): d 23.73, 23.77, 39.81, 57.58, 71.70, 71.77,
120.00, 127.50, 127.84, 128.05, 128.43, 128.69, 129.59, 129.68,
133.41, 136.58, 137.302, 137.38, 126.40, 127.02, 170.80. HRMS:
calcd for [C29H36N2O4P]+: 507.2413; found: 507.2424.
4.2.6. Capase-8 and caspase-9 assays
The untreated and compound-treated cells for 0, 2, 4, 8, 14, and
24 h were collected by trypsinization, rinsed twice with PBS, resus-
pended in lysis buffer (caspase-8 and caspase-9 activity assay kit),
and incubated on ice for 15 min. The lysate was centrifuged at 4 °C
for 15 min at 20,000g for 15 min. The supernatant was assayed for
protein by the Bradford method with BSA as a standard, and 0.1 mg
was used to assay caspase activity with Ac-IETD-pNA and Ac-
LEHD-pNA as substrate for caspase-8 and caspase-9, respectively,
Abs405 nm caused by the production of pNA were continuously re-
corded by Multimode Detector DTX880 (Beckman Coulter) after
incubation for 2 h at 37 °C incubator
4.1.6.20. Compound 9j: 4-(N-O,O0-diisopropylphosphoryl-
amido-E-stilbene.
Yield 78%; oil; 31P NMR (CDCl3, 202 MHz):
L-Pro)
d 7.87. 1H NMR (CDCl3, 300 MHz): d 1.31 (m, 12H), 1.96 (m,4H),
2.52 (m, 1H), 3.25 (m, 2H), 4.27 (m, 1H), 4.63 (m, 2H), 7.03 (d, 1H,
J13 = 16.50 Hz), 7.07 (d, 1H, J13 = 16.50 Hz), 7.24 (m, 1H), 7.34 (m,
2H), 7.46 (m, 2H), 7.49 (m, 2H), 7.59 (m, 2H), 9.59 (s, 1H). 13C NMR
(CDCl3, 125 MHz): d 23.92, 23.99, 25.59, 29.01, 47.77, 61.73, 71.75,
71.84, 119.46, 127.40, 127.56, 128.16, 128.66, 132.99, 137.47,
137.89, 126.37, 127.09, 170.80. HRMS: calcd for [C25H34N2O4P]+:
457.2257; found: 457.2270.
4.2.7. ROS generation assays
The untreated and compound-treated cells for 0, 2, 4, 6, 8, 13, 19,
24, and 30 h, were collected by trypsinization, precipitated through
centrifugation at 1000g for 5 min, and washed twice with PBS. Intra-
cellular ROS production was measured by using a fluorescent dye
DCFH-DA, which can be converted to DCFH by esterases when the
cells take it up. The ROS of cells can oxidize the DCFH to DCF which
can be detected by FACS. The cells were incubated with DCFH-DA
4.2. Biology
4.2.1. Cell culture
Cell lines CNE-1 and CNE-2 were cultured in DMEM, with 10%
fetal bovine serum (FBS), 100 lg/ml penicillin, and 100 lg/ml
(10
lM) at 37 °C for 30 min and then analyzed with the FACS.
streptomycin in humidified air at 37 °C with 5% CO2.
Acknowledgments
4.2.2. Cell-growth inhibition assay
The CNE-1/CNE-2 cells were seeded in 96-well microtiter plate
and treated with the synthesized compounds at different concen-
The authors thank the financial supports from the Ministry of
Science and Technology of China (2007AA02Z160), the Chinese Na-
tional Natural Science Foundation (20672068, 20872077, and
90813013), the Ministry of Education (20070003077).
trations, ranging from 0.5 to 100
treatment, the cells were incubated with MTT (5 mg/ml) in 15
for 4 h, The formazan precipitate was dissolved in 100 L DMSO
lM for 24, 48, and 72 h after
lL
l
and the absorbance at 595 nm of each well was measured by Mul-
Supplementary data
timode Detector DTX880 (Beckman Coulter).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.022.
4.2.3. DNA fragmentation assay
DNA was prepared from the untreated and compound-treated
cells by standard digestion and organic extraction. Extracted DNA
was separated by electrophoresis in 2% agarose gel and visualized
with ethidium bromide staining.
References and notes
1. Lyons, M.; Yu, C.; Toma, R. B.; Cho, S. Y.; Reiboldt, W.; Lee, J.; Breemen, R. B. V. J.
Agric. Food Chem. 2003, 51, 5867.
2. Todaro, A.; Palmeri, R.; Barbagallo, R. N.; Pifferi, P. G.; Spagna, G. Food Chem.
2008, 107, 1570.
4.2.4. PARP cleavage assay
Cells treated for 24 h with different analogues (8d, 8e, 8j, and
3. Burns, J.; Yokota, T.; Ashihara, H.; Lean, M. J.; Crozier, A. J. Agric. Food Chem.
2002, 50, 3337.
4. Sanders, T. H.; McMichael, R. W., Jr.; Hendrix, K. W. J. Agric. Food Chem. 2000, 48,
1243.
5. Ibern-Gomez, M.; Roig-Perez, S.; Lamuela-Raventos, R. M.; Torre-Boronat, M. C.
d. l. J. Agric. Food Chem. 2000, 48, 6352.
6. Baur, J. A.; Pearson, K. J.; Price, N. L.; Jamieson, H. A.; Lerin, C.; Kalra, A.; Prabhu,
V. V.; Allard, J. S.; Lopez-Lluch, G.; Lewis, K.; Pistell, P. J.; Poosala, S.; Becker, K.
G.; Boss, O.; Gwinn, D.; Wang, M.; Ramaswamy, S.; Fishbein, K. W.; Spencer, R.
G.; Lakatta, E. G.; Couteur, D. L.; Shaw, R. J.; Navas, P.; Puigserver, P.; Ingram, D.
K.; Cabo, R. d.; Sinclair, D. A. Nature 2006, 444, 337.
9d) at 50 lM and without treated, were lysed for 30 min on ice ly-
sis buffer (50 mM Tris–base, 1.0 mM EDTA, 150 mM NaCl, 0.1%
SDS, 1% Triton X-100, 1% sodium deoxycholate, and 1 mM PMSF).
Cytosolic proteins were obtained by centrifugation at 15,000g for
15 min of extracts of cells. Proteins (25 lg) were separated on
12% SDS–polyacrylamide minigel and transferred to nitrocellulose
membranes. Membranes were incubated in blocking solution (5%
non-fat dry milk in 0.05% Tween 20 in 50 mM Tris, 150 mM NaCl)
and anti-PARP antibody (1:1000 dilution in blocking solution).
PARP, intact protein and cleaved fragment were visualized using
an enhanced chemiluminescence detection system (Bio-Rad). The
expression of b-actin was used as a normalization control for pro-
tein loading.
7. Frekmont, L. Life Sci. 2000, 66, 663.
8. Bureau, G.; Longpre, F.; Martinoli, M.-G. J. Neurosci. Res. 2008, 86, 403.
9. Lagouge, M.; Argmann, C.; Gerhart-Hines, Z.; Meziane, H.; Lerin, C.; Daussin, F.;
Messadeq, N.; Milne, J.; Lambert, P.; Elliott, P.; Geny, B.; Laakso, M.; Puigserver,
P.; Auwerx, J. Cell 2006, 127, 1109.
10. Jang, M.; Cai, L.; Udeani, G. O.; Slowing, K. V.; Thomas, C. F.; Beecher, C. W. W.;
Fong, H. H. S.; Farnsworth, N. R.; Kinghorn, A. D.; Mehta, R. G.; Moon, R. C.;
Pezzuto, J. M. Science 1997, 275, 218.
11. Minutolo, F.; Sala, G.; Bagnacani, A.; Bertini, S.; Carboni, I.; Placanica, G.; Prota,
G.; Rapposelli, S.; Sacchi, N.; Macchia, M.; Ghidoni, R. J. Med. Chem. 2005, 48,
6783.
4.2.5. Flow cytometric analysis of apoptosis
Untreated and drug-treated cells were collected by centrifuga-
tion and washed twice with ice-cold PBS. The cells were fixed with

H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 10013–10021
10021
12. Cardilea, V.; Lombardoa, L.; Spataforab, C.; Tringali, C. Bioorg. Chem. 2005, 33, 22.
13. Kuo, P.-L.; Chiang, L.-C.; Lin, C.-C. Life Sci. 2002, 72, 23.
14. Pettit, G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R. K.; Chapuis, J.-C.;
Schmidt, J. M. J. Med. Chem. 2002, 45, 2534.
15. Park, C.-S.; Lee, Y.-C.; Kim, J.-D.; Kim, H.-M.; Kim, C.-H. Vasc. Pharmacol. 2004,
40, 279.
23. Rimando, A. M.; Cuendet, M.; Desmarchlier, C.; Mehta, R. G.; Pezzuto, J. M.;
Duke, S. O. J. Agric. Food Chem. 2002, 50, 3453.
24. Tolomeo, M.; Grimaudo, S.; Cristina, A. D.; Roberti, M.; Pizzirani, D.; Meli, M.;
Dusonchet, L.; Gebbia, N.; Abbadessa, V.; Crosta, L.; Barucchello, R.; Grisolia, G.;
Invidiata, F.; Simoni, D. Int. J. Biochem. Cell B 2005, 37, 1709.
25. Zhang, W.; Go, M. L. Eur. J. Med. Chem. 2007, 42, 841.
16. Stivala, L. A.; Savio, M.; Carafoli, F.; Perucca, P.; Bianchi, L.; Maga, G.; Forti, L.;
Pagnoni, U. M.; Albinii, A.; Prosperi, E.; Vannini, V. J. Biol. Chem. 2001, 276, 22586.
17. Roberti, M.; Pizzirani, D.; Simoni, D.; Rondanin, R.; Baruchello, R.; Bonora, C.;
Buscemi, F.; Grimaudo, S.; Tolomeo, M. J. Med. Chem. 2003, 46, 3546.
18. Lion, C. J.; Matthews, C. S.; Stevens, M. F. G.; Westwell, A. D. J. Med. Chem. 2005,
48, 1292.
26. Galardy, R. E.; Grobelny, D. J. Med. Chem. 1985, 28, 1422.
27. Niu, Y. L.; Du, W.; Jiang, Y. Y.; Zhao, Y. F. Phosphorus, Sulfur Silicon Relat. Elem.
2002, 177, 2083.
28. Yang, J.; Jiang, Y. Y.; Liu, F.; Zhao, Y. F. Protein Peptide Lett. 2006, 13, 129.
29. Ding, G. Y.; Liu, F.; Yang, T.; Jiang, Y. Y.; Fu, H.; Zhao, Y. F. Bioorg. Med. Chem.
2006, 14, 3766.
19. Reddy, M. V. R.; Mallireddigari, M. R.; Cosenza, S. C.; Pallela, V. R.; Iqbal, N. M.;
Robell, K. A.; Kang, A. D.; Reddy, E. P. J. Med. Chem. 2008, 51, 86.
20. Gao, M.; Wang, M.; Miller, K. D.; Sledge, G. W.; Hutchins, G. D.; Zheng, Q.-H.
Bioorg. Med. Chem. Lett. 2006, 16, 5767.
21. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.;
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.
30. Pinney, K. G.; Mejia, M. P.; Villalobos, V. M.; Rosenquist, B. E.; Pettit, G. R.;
Verdier-Pinard, P.; Hamel, E. Bioorg. Med. Chem. 2000, 8, 2417.
31. Minutolo, F.; Sala, G.; Bagnacani, A.; Bertini, S.; Carboni, I.; Placanica, G.; Prota, G.;
Rapposelli, S.; Sacchi, N.; Macchia, M.; Ghidoni, R. J. Med. Chem. 2005, 48, 6783.
32. Kothakota, S.; Azuma, T.; Reihard, C.; Klippel, A.; Tang, J.; Chu, K.; McGarry, T. J.;
Kirschner, M. W.; Koths, K.; Kwiatkowski, D. J. Science 1997, 278, 294.
33. Fesik, S. W. Cell 2000, 103, 273.
22. Pan, M. H.; Chang, Y. H.; Brdmaev, V.; Nagabhushanam, K.; Ho, C.-T. J. Agric.
Food Chem. 2007, 55, 7777.
34. Simoni, D.; Tolomeo, M. Curr. Pharm. Des. 2001, 7, 1823.